The investigators in this Shared Instrument Grant (SIG) application at New Jersey Medical School request funds for purchase of a state-of-the-art Amnis ImageStreamX Mark II imaging flow cytometer to replace our outdated, first-generation ImageStream 100 that was purchased in late 2007 with an SIG awarded to the PI of the current application. The ImageStreamX Mark II will be housed and administrated through the Flow Cytometry and Immunology Core Laboratory at NJMS, which has strong institutional support from NJMS. Imaging flow cytometry provides the high-throughput content of traditional flow cytometry coupled with the ability to carry-out image analysis on cell populations in flow. This technology allows for analysis, for example, of nuclear translocation, co-localization studies, intracellular pathogen tracking, phagocytosis, FRET, and apoptosis in populations of cells. We are requesting that the ImageStreamX Mark II be equipped with four excitation lasers: 488, 405, 561 and 592nm lasers, plus the standard 785nm SSC laser. In addition, we request that the instrument be equipped with 12 imaging channels, the Multi-Mag option to provide flexible 20X, 40X and 60X imaging capabilities, an extended depth-of-field element, and a 96-well plate auto-sampler. Finally, we request a stand-alone work station for data analysis. The requested ImageStreamX Mark II has multiple improvements over the first generation instrument: most importantly, the ImageStreamX Mark II as requested will allow for up to 10-fluorochomes to be used in a single sample and is up to 20X faster than the original ImageStream 100. The ability to simultaneously evaluate 10 markers in imaging flow dramatically extends the capability of the instrument compared to the four colors with our original instrument and allows investigators to adapt multi-color flow panels used for traditional flow and cell sorting to the ImageStreamX Mark II. These improvements to the instrumentation, along with the Multi-Mag element make the ImageStreamX Mark II a much more versatile instrument that can be used to carry-out sophisticated, multi-color flow cytometric analysis, even on very rare cells. The SIG investigators represent a group of highly-productive scientists from diverse departments at the New Jersey Medical School, almost all of whom have NIH R01 funding. Many of the SIG investigators have effectively utilized our existing ImageStream 100 and have published papers and obtained grants with ImageStream data, but have found the need for more sophisticated imaging flow cytometry. There are 8 major and 5 major users for this SIG application. While many are immunologists, the SIG investigators also include a neuroscientist, a cell physiologist, a microbiologist and biochemists. Thus, the ImageStreamX Mark II is expected to provide valuable imaging flow cytometry capabilities across a wide diversity of disciplines. This instrument will enhance the productivity of the SIG investigators and will also provide valuable opportunities for pre- and post-doctoral trainees.